In addition to recombinant DNA technology to provide proteins, the chemical synthesis of proteins has dramatically contributed to the exploration of the relationship of protein structure to function. Moreover, with the rapid emergence of peptides of middle size (between 20 and 100 amino acids) as therapeutics, synthetic peptide chemistry has been flourishing more than ever. Merrifield's linear solid phase peptide synthesis (SPPS) has provided a general tool to prepare polypeptides. However, to chemically synthesize a large polypeptide (>50 amino acids) using linear solid phase peptide synthesis is very costly and sometimes even impossible. Thus, the method to achieve a convergent synthesis of polypeptides becomes critical. Using less or no protecting groups and the efficiency of the coupling step are the critical issues in the development of a convergent synthesis. Use of unprotected peptides for chemical manipulation circumvents the difficulty inherent to classical peptide coupling reactions derived from limited solubility, thus increasing the coupling yield, and leading to the easy purification and characterization. The key issue to develop peptide coupling methods using unprotected peptide segments is the availability of a chemoselective reaction to specifically and unambiguously join peptides through C-terminus of a peptide and N-terminus of a second peptide. Using a chemoselective reaction to join two peptide segments through formation of an unnatural (i.e. non-peptide) backbone structure at the ligation site has permitted the facile preparation of a wide range of backbone-modified artificial peptides and proteins. However, in order to achieve native chemical ligation (ligating two peptide segments through natural peptide bonds), chemical method is very limited. Using unprotected peptides, forming natural peptidic bonds at the ligation site and resulting in no or little epimerization of the formed peptide bond are characteristics of a practical native chemical ligation.
Clearly, cysteine based native chemical ligation (NCL), developed by Kent and coworkers, meets these criteria, and it has become no doubt the most powerful method in synthetic peptide chemistry (Dawson P. E.; Muir, T. R.; Clarklewis, I.; Kent, S. B. H. Science, 1994, 266, 776-779, “synthesis of proteins by native chemical ligation”). This method enables a convergent synthesis of larger size peptides and even proteins. The repertoire of NCL has widely been expanded to various aspects in chemistry and biology over the past 15 years. Cysteine based NCL features a thio capture between an N-terminal cysteine and a C-terminal thioester, as a transthioesterification step which is highly chemoselective, followed by a rapid S→N acyl transfer to afford a natural Xaa-Cys peptidic linkage (Xaa represents any amino acid). Its efficiency, easy operation and chemoselectivity (in presence of any unprotected amino acid) are very attractive to its users/practitioners, thus cysteine based NCL has been widely used for chemically synthesizing many proteins (>100 amino acids). That the cysteine based NCL achieves the chemoselectivity lies in that the N-terminus cysteine can differentiate itself from other inner unprotected amino acid functional groups with its bifunctionalities: a 1,2-mercapto-amine. Moreover, the capture-rearrangement chemical ligation does not involve activating the carboxyl group, thus native chemical ligation overcomes the racemerization problem of the conventional segment condensation method.
However, the rare presence of the cysteine residue (1.4% content in proteins) has limited the utility of the above NCL. To address this issue, people have extensively searched for alternative native chemical ligation methods at other amino acid sites. By far, all alternatives recently developed follow the line of cysteine based native chemical ligation, relying on the thio-capture-rearrangement strategy. Many efforts are focused on introducing β or γ-thio group into natural amino acids. After the cysteine-like native chemical ligation, the thio group of the ligated product is removed to achieve a ligation at the corresponding amino acid. Other efforts are directed to the development of chemical manipulation conditions to convert the cysteine-NCL product into other forms of amino acids, i.e. desulfurization of cysteine to give alanine. All these methods have expanded the arsenal for peptide/protein ligation and have been used to prepare various (glyco)peptide/proteins at a very complicated level (Offer, J.; Boddy, C. N. C.; Dawson, P. E. J. Am. Chem. Soc., 2002, 124, 4642-4646, “Extending synthetic access to proteins with a removable acyl transfer auxiliary”). However, the practice of these methods requires either sophisticated chemistry or syntheses of unnatural amino acids, therefore, they are not as generally used as cysteine-based NCL. Moreover, these methods all affect unprotected cysteine residues in addition to the cysteine residue at the ligation site. The chemoselective ligation using user-friendly conditions at other amino acid sites has yet to be discovered.